Chemokines constitute a family of small pro-inflammatory cytokines with leukocyte chemotactic and activating properties. Depending on the position of the first conserved cysteines, the chemokine family can be divided in C—C, C—X—C and C—X3—C chemokines (Baggiolini M. et al., Adv Immunol. 1994, 55:97-179; Baggiolini M. et al., Annu Rev Immunol. 1997,15:675-705; Taub D. et al., Cytokine Growth Factor Rev. 1996,7(4):355-76).
Many C—X—C chemokines such as interleukin-8 (IL-8) are chemotactic for neutrophils, while C—C chemokines are active on a variety of leukocytes including monocytes, lymphocytes, eosinophils, basophils, NK cells and dendritic cells.
The NH2-terminal domain of chemokines is involved in receptor binding and NH2-terminal processing can either activate chemokines or render chemokines completely inactive.
N-terminal variants of synthetical C—C chemokines have been tested for their activity as inhibitors or antagonists of the naturally occurring forms. MCP-1, MCP-3 and RANTES missing the 8 to 9 NH2-terminal amino acids are inactive on monocytes and are useful as receptor antagonists (Gong J H et al., J Exp Med. 1995,181(2):631-40 and Gong J H et al., J Biol Chem. 1996, 271(18):10521-7).
Extension of RANTES with one methionine results in almost complete inactivation of the molecule and Met-RANTES behaves as an antagonist for the authentic one (Proudfoot A E et al., J Biol Chem. Feb. 2, 1996;271 (5):2599-603).
WO 99/16877 relates to amino-terminally truncated RANTES, lacking NH2-terminal amino acids corresponding to amino acid residues 1, 1-2, 1-3 or 1-4 of the naturally-occurring RANTES and having chemokine antagonistic activity, as well as cDNA sequences encoding them, their use in therapy and/or in diagnosis of the diseases, in which an antagonistic activity of the chemokine effects is required. RANTES (3-68) is the preferred truncated chemokine antagonist.
Even if the chemoattractant activity of RANTES and of CC chemokines in general has been studied mainly in connection with the specific cell membrane receptors, RANTES can interact also with Glycosaminoglycans (GAGs), highly variable, branched sugar groups added post-translationally to several proteins, generically called proteoglycans (PGs). Such proteins are present on cell membrane, in the extracellular matrix and in the blood steam, where isolated GAGs can also be present.
The interaction with GAGs is a feature common to many cel-signaling soluble molecules (interleukins, growth factors). PGs, or isolated GAGs, can form a complex with soluble molecules, probably at the scope to protect this molecule from proteolysis in the extracellular environment It has been also proposed that GAGs may help the correct presentation of cell signaling molecules to their specific receptor and, eventually, also the modulation of target cell activation.
In the case of chemokines, the concentration into immobilized gradients at the site of inflammation and, consequently, the interaction with cell receptors and their activation state seem to be modulated by the different forms of GAGs (Hoogewerf A J et al., Biochemistry 1997, 36(44):13570-8). Therefore, it has been suggested that the modulation of the such interactions may represent a therapeutic approach in inflammatory disease (Schwarz M K and Wells TN, Curr Opin Chem Biol. 1999, 3(4):407-17) and in HIV infection (Bums J M et al., Proc Natl Acad Sci U.S.A. 1999, 96(25):14499-504).
The structural requirements and functional effects of GAG-RANTES interaction have been studied in various models. RANTES binds GAGs on human umbilical vein endothelial cells (HUVECs) at micromolar concentrations with an affinity and a specificity higher then other chemokines, like MCP-1, IL-8, or MIP-1 alpha. Such interaction appears to be not simply electrostatic but also depending by other parameters like length and N- and O-sulfation of the GAGs (Kuschert G S et al., Biochemistry 1999, 38(39):12959-68). GAG-defective cell lines still can bind chemokines but the presence of cell surface GAGs greatly enhances their activity on the receptors when they are at low concentrations (Ali S et al., J Biol Chem 2000, 275(16):11721-7). Other experiments showed that GAGs, heparin sulphate in particular, facilitate the interaction of RANTES with the cell surface of macrophages and the consequent inhibition of HIV infection, a result consistent with the well-known resistance of these cells, poorly expressing heparin sulphate, to antiviral effects of RANTES (Oravecz T, et al., J Immunol. 1997,159(9):4587-92).
Soluble GAGs compete with cell membrane GAGs, and they can act as specific inhibitors of RANTES-induced activation surface (Appay V, et al.; Int Immunol 2000, 12(8):11737-82), or as suppressor HIV infection (Bums J M, et al.; Proc Natl Acad Sci U.S.A. 1999, 96(25):14499-504).
Some structure-function studies tried to identify the RANTES domain responsible of the interaction with GAGs, since the traditional consensus sequence (BBXB, where B is a basic residue and X can be any residue) is too generic. An epitope-mapping study was performed by using a monoclonal antibody, raised against recombinant human RANTES, capable to block both the antiviral effects and the mobilization of intracellular calcium mediated by RANTES (Bums J M et al., J. Exp . Med. 1998, 188(10):1917-27). This approach allowed to define the residues 55-66 as necessary both for such activities and for GAG interaction, arguing that GAGs interaction may have a complementary or distinct function from the one mediated by canonical receptors, as also suggested in a study on RANTES variants having altered aggregation properties (Appay V et al., J Biol Chem 1999, 274(39):27505-12).
The region 55-66, which represents the C-terminal alpha-helical segment, is homologous to the GAG-binding domain of other chemokines, like IL-8 (Witt DP and Lander AD, Curr. Biol. 1994, 4(5):394-400), and contains a cationic site containing lysine and arginine (KKWVR) (SEQ ID NO: 39). Such binding region is distinct from the binding site for cell receptors, which is located at the N-terminus (Pakianathan DR et al., Biochemistry 1997, 36(32): 9642-8), and contains some residues involved in the aggregation of RANTES monomers, even though such disaggregating mutations seem not to affect the interaction with GAGs (Czaplewski L. G. et al., J. Biol. Chem. 1999, 274(23): 1607784; WO 98/13495).
RANTES contains another cationic site (RKNR) at residues 44-47 which is conserved in the GAG binding domain of other chemokines, like MIP-1α (Koopmann W and Krangel MS; J. Biol. Chem. 1997, 272(15):10103-9) and MIP-1β (Koopmann W et al., J Immunol. 1999, 163(4):2120-7).
Human RANTES variants containing single mutations in these cationic sites have been disclosed as RANTES antagonists having potential therapeutic applications in the treatment of HIV infection and inflammatory or allergic diseases (WO 99/33989).
It has also been disclosed that only a triple mutant of RANTES, in which three residues at positions 44, 45 and 47 have been substituted with Alanine, has lost the GAG-binding ability (A Proudfoot et al., Chemokine Gordon Conference, Session I, Jul. 24, 2000, personal communication).